The research in this proposal is concerned with genetic and biochemical mechanisms responsible for the initiation of replication and partitioning of the broad-host-range plasmid RK2 in Escherichia coli and distantly related Gram-negative bacteria. Plasmid RK2 specifies resistance to the antibiotics ampicillin, tetracycline and kanamycin, and will replicate and is stably maintained in a wide range of Gram-negative bacteria. Mechanisms of plasmid replication initiation and segregation to daughter cells will be investigated in E. coli, Pseudomonas putida and Pseudomonas aeruginosa using biochemical, genetic and cytological techniques. RK2 encodes a replication initiation protein (TrfA) and a replication origin that has as its main features 17 base pair repeats (iterons) that are bound by the TrfA protein, four DnaA boxes, and an A+T rich sequence that contains four 13-mer sequences. The plasmid also contains two regions, including the par operons, which are involved in stable maintenance. A major thrust of the proposed research is understanding the unique properties of this plasmid that account for its ability to initiate its replication and faithfully partition itself during cell division in a wide range of bacteria. To this end, the activities of the key host proteins DnaA and DnaB of E. coli, P. aeruginosa, and P. putida along with the plasmid specific initiation protein in the initiation of replication of RK2 and narrow-host-range plasmids P1 and F will be determined. In addition, the activities of these various host proteins (along with the DnaC protein of E. coli) at the chromosomal replication origins of E. coli and the two Pseudomonas strains will be compared. Both FISH and GFP-tagging techniques will be used to localize the RK2 plasmid in wild-type and mutant E. coli strains and in bacteria distantly related to E. coli. GFP-tagging of RK2 and of specific replication proteins will be used for time-lapse analysis of the dynamic movement of this plasmid and replication proteins during cell growth and division. These various studies should contribute to our understanding of the fundamental processes of initiation of DNA replication, DNA segregation, and the dissemination of antibiotic resistance in bacteria.